1. Field of the Invention
The invention is generally related to the fields of pharmacology and immunology. More specifically, the invention is directed to novel compositions of matter for selectively activating T cells (PHA-blasts), and having reduced activation of Natural Killer (“NK”) cells. The novel compositions include variants of the cytokine family, and in particular human Interleukin-2 (“IL-2”).
2. Description of Related Art
Interleukin 2 (IL-2) is a potent immune stimulator, activating diverse cells of the immune system, including T cells, B cells, and monocytes. IL-2 is also a potent and critical growth factor of T cells. It was by virtue of these activities that IL-2 was tested for its ability to treat cancer. Human IL-2 is an-FDA approved drug for the treatment of metastatic renal carcinoma and metastatic melanoma. The use of IL-2 in eligible patients is restricted due to the severe toxicity associated with IL-2 therapy; it is estimated that at best only 20% of eligible patients actually receive therapy. The toxicities associated with IL-2 therapy include severe fever, nausea, vomiting, vascular leak and serious hypotension. Despite these toxicities, however, IL-2 is effective for its approved indications (˜17% objective response rate).
Although the structure/function analysis of mouse IL-2 has been extensive (Zurawski, S. M. and Zurawski, G. (1989) Embo J 8: 2583-90; Zurawski, S. M. et. al., (1990) Embo J 9: 3899-905; Zurawski, G. (1991). Trends Biotechnol 9:250-7; Zurawski, S. M. and Zurawski. G. (1992) Embo J 11: 3905-10. Zurawski, et. al., EMBO J, 12 5113-5119 (1993)), only limited analysis of human IL-2 has occurred. Most studies with human IL-2 muteins have been performed on murine cells; however, limited studies have occurred using human PHA-blasts, which express the high affinity IL-2 receptor IL-2Rαβγ. These studies using PHA-blasts confirmed the importance of the residues Asp-20 and the D-helix of human IL-2. It has been shown that position Asp-20 and Gln-126 of human IL-2 are the primary residues responsible for interaction with the IL-2 receptor β-and γ-subunits, respectively (reviewed in Thèze, et. al., Immunol. Today, 17, 481-486 (1996)) Although residues in the C-helix of mouse IL-2 have been shown to be involved in interaction with mouse IL-2Rβ (Zurawski, et. al., EMBO J, 12 5113-5119 (1993)), the equivalent residues in human IL-2 have not been shown to have the same properties (these residues in human IL-2 would be Asp-84 and Asn-88). Because significant species specificity is shown between human and mouse IL-2 (human IL-2 exhibits ˜100-fold reduced activity in murine systems), it is difficult to predict whether the same type of interactions are occurring within species boundaries. No studies are known using cells expressing only the human intermediate affinity receptor IL-2Rβγ.
Some human IL-2 muteins have been examined for their activity on human PHA blasts (Xu, et. al., Eur. Cytokine Netw, 6, 2237-244 (1995)). Muteins containing substitutions of Asp-20 with Leucine (D20L), as well as Arginine, Asparagine, and Lysine, were shown to have severe defects in their ability to induce proliferation of PHA-blasts. Thus, this art teaches that substitution of Asp20 will result in muteins of compromised activity. Additionally, Xu, et al states that to date (1995) no useful IL-2 muteins have been identified for either clinical or research applications.
The human IL-2 Q126D mutein generated by Buchli and Ciardelli, Arch. Biochem. Biophys, 307(2): 411-415, (1993) showed significantly compromised activity in both potency and agonism, in human T cell assays, it exhibited ˜1,000-fold less activity than IL-2 and behaved as a partial agonist. In murine T cell assays, the mutein was nearly inactive. Both cell lines tested expressed the high affinity form of the IL-2 receptor. On both cell types, Q126D displayed the ability to antagonize IL-2-mediated activity, although only partially in the human T cell assay.
Zhi-yong, W., et al., Acta Biochimica et Biophysica Sinica 25(5):558-560 (September 1993) performed substitution experiments on IL-2 at positions 62, 69, 99 and 126, demonstrating a 20-fold and 30-fold reduction in activity as compared to wt IL-2, with 62-Leu-IL-2 and 126-Asp-IL-2, respectively, in a mouse T-cell assay (CTLL-2). However, there is no teaching or suggestion that substitutions at position 126 may confer T-cell selective activity over NK cells, or indicate whether such changes would have a similar effect on human T cells.
Collins, L., et al., PNAS USA 85:7709-7713 (1988) reported that substition of Asp at position 20 with either Asn (D20N) or Lys (D20K) resulted in a loss of binding ˜100- to 1,000-fold relative to human IL-2 for both the high (IL-2Rαβγ, termed p55/p70 in Collins, et al) and the intermediate affinity receptor (IL-2Rβγ, termed “p70” in Collins, et al). Binding to IL-2Rα appeared unaffected for both mutant proteins. This work teaches that disruption of binding to the intermediate affinity IL-2 receptor (IL-2Rβγ) will also lead to disruption of binding to the high affinity IL-2 receptor (IL-2Rαβγ), suggesting that differential binding or activation between IL-2Rβγ or IL-2Rαβγ is not achievable by substitution of Asp at position 20.
Berndt, W. G., et al., Biochemistry 33(21):6571-6577 (1994) used combinatorial cassette mutagenesis to simultaneously mutate positions 17-21 in native IL-2, which are suspected to interact with the intermediate affinity IL-2 receptor. Out of 2610 screened clones, only 42 were active. They found that positions 20 and 21 were of primary importance for biological activity. There is no suggestion or teaching of individual substitutions except for L21V.
U.S. Pat. No. 5,229,109 (Grimm, et al.) allegedly disclose low toxicity IL-2 analogs for use in immunotherapy and cancer treatment. The properties of two IL-2 analogs with substitutions at positions Arg38 (to Alanine) and Phe42 (to Lysine) were analyzed and compared to those of native IL-2. The analogs were found to be able to maintain their ability to bind to the intermediate IL-2 receptor, while binding only minimally to the so-called “high affinity” receptor. At this time the intermediate affinity receptor was thought to consist only of p75 (IL-2Rβ), and the high affinity receptor was thought to consist only of p55+p75 receptor complex (IL-2Rαβ). The analogs also maintained their ability to stimulate peripheral blood mononuclear cells to generate lymphokine activated killing (LAK). Notably, IL-1β and TNFα secretion were significantly reduced in response to the analogues, as compared to the native IL-2 molecule. The amino acid residues described in this patent are those that would interact specifically with IL-2Rα (p55); elimination of interactions with IL-2Rα would result in reduced activity on high affinity IL-2 receptor bearing cells, and would not affect activity to intermediate affinity IL-2 receptor bearing cells. Thus, generation of LAK cells (which are thought to be derived from NK cells) should be maintained. The muteins described herein are directed to amino acid residue positions 20, 88, and 126; these positions are thought to interact specifically with IL-2Rβ (p75; positions 20 and 88), and IL-2Rγ (not known at the time of the filing of the Grimm, et al patent; positions 126). As a consequence, interactions with IL-2Rα remain unchanged. Mechanistically, the muteins described by Grimm, et al, will have decreased interactions with the IL-2 high affinity receptor, but should have no effect on the IL-2 intermediate affinity receptor; the muteins described herein have the opposite characteristic, possessing a pronounced defect in their ability to interact with the IL-2 intermediate affinity receptor IL-2Rβγ, and show little or no defect in functional interactions with the IL-2 high affinity receptor, IL-2Rαβγ.
U.S. Pat. No. 5,206,344, (Goodson et al.) discloses muteins of IL-2 in which one of the amino acids of the mature native sequence of IL-2 is replaced by a cysteine residue, which are then prepared and conjugated through the replaced cysteine residue to a polymer selected from polyethylene glycol homopolymers or polyoxyethylated polyols, wherein the homopolymers are unsubstituted or substituted at one end with an alkyl group. These muteins are made via host expression of mutant genes encoding the muteins that have been changed from the genes for the parent proteins by site-directed mutagenesis. In addition, other species of IL-2 may be conjugated via the cysteine residue at position 125 of the mature IL-2 protein that is not necessary for the biological activity of the IL-2. There is no disclosure of mutations that confer reduced toxicity.
U.S. Pat. No. 4,959,314 (Lin et al.) discloses muteins of biologically active proteins such as IFN-β and IL-2 in which cysteine residues that are not essential to biological activity have been deleted or replaced with other amino acids to eliminate sites for intermolecular crosslinking or incorrect intramolecular disulfide bridge formation. These muteins are made via bacterial expression of mutant genes that encode the muteins that have been synthesized from the genes for the parent proteins by oligonucleotide-directed mutagenesis. There is no disclosure of mutations that confer reduced toxicity.
U.S. Pat. No. 5,116,943 (Halenbeck et al.) discloses that a biologically active reference therapeutic protein is protected against oxidation by a method involving substituting a conservative amino acid for each methionyl residue susceptible to chloramine T or peroxide oxidation, wherein additional, nonsusceptible methionyl residues are not so substituted. The oxidation-resistant mutein so produced is preferably a human mutein of interleukin-2 or interferon-β, and the conservative amino acid is most preferably alanine. There is no disclosure of mutations that confer reduced toxicity.
U.S. Pat. No. 4,853,332 (Mark et al.) is directed to IFN-γ and IL-2 muteins in which cysteine residues that are not essential to biological activity have been deleted or replaced with other amino acids to eliminate sites for intermolecular cross-linking or incorrect intramolecular disulfide bridge formation. The patent discloses that a substitution in IL-2 at cysteine 125 to serine results in a mutein with activity comparable to native IL-2.
U.S. Pat. No. 5,696,234 (Zurawski et al.) is directed to muteins of mammalian cytokines, and methods for screening for agonists and antagonists of mammalian cytokines. In particular, human IL-2 double mutein P82A/Q126D is demonstrated as having antagonist activity on murine Baf3 cells cotransfected with human α and β IL-2R subunits. Little agonist activity is shown. Also, murine IL-2 muteins are shown to exhibit partial agonist and antagonist activity on HT2 cells, in particular Q141D, Q141K, Q141V, and Q141L. No mutein activities are described that show or suggest a selective agonist effect for mutations at positions 20, 88 and 126.
Zurawski, et al, describe murine IL-2 muteins that have properties of being active on cells expressing IL-2Rαβγ but inactive on cells expressing IL-2Rβγ (Zurawski, G., Trends Biotechnol, 9: 250-257 (1991); Zurawski, S. M. and Zurawski, G. Embo. J. 11:3905-3910 (1992)). The murine IL-2 muteins that exhibited these properties had the substitutions Asp-34 to Ser or Thr, and Gln-141 to Lys. Asp-34 and Gln-141 of mouse IL-2 appear equivalent to Asp-20 and Gln-141 of human IL-2, respectively. Although these references refer to “selective agonist” IL-2 muteins, they do not describe their potential as being less toxic, but rather as being potential antagonists of endogenous IL-2.
EP 0267 795 A2 (Zurawski et al.) discloses a variety of mouse IL-2 muteins throughout the sequence, including some containing deletions and/or substitutions within the first thirty N-terminal amino acid residues which are biologically competent, but it does not include, discuss or suggest the amino acid substitutions disclosed here at the equivalent mouse IL-2 residues. There exists a need for an improved IL-2 molecule which has reduced toxicity and is more generally tolerated.
Taniguchi et al., U.S. Pat. No. 4,738,927 is directed to a recombinant DNA which comprises a recombinant DNA encoding a polypeptide which possesses a biological activity of IL-2, wherein said activity is promotion of growth of a cytotoxic T-lymphocyte cell line, and a vector DNA capable of propagating in a prokaryotic or eukaryotic cell, the coding sequence of said gene being located at a position downstream of a promoter sequence, wherein said polypeptide has 132 to 134 amino acids in total in the amino acid sequence of the polypeptide. It also describes a gene, recombinant DNA vectors, host cells, and methods of recombinantly producing native IL-2. Taniguchi et al. do not describe any variants or muteins, and do not teach which positions in the protein are responsible for signaling or binding activity.
It is clear that IL-2 muteins that do not exhibit the dose-limiting toxicity of the prior art recombinant IL-2 muteins are needed to take therapeutic advantage of the potential of this cytokine.